During the previous funding period, we defined novel roles for GTPases and their regulatory proteins in various aspects of Golgi and endosomal vesicle function. The experiments proposed in this application result directly from these new insights, and will provide biochemical data linking defective endosomal acidification to aberrant vesicle coat recruitment and abnormal proximal tubule absorptive function leading to disease at the whole animal level. Our specific aims are: 1) To determine the molecular mechanism of Arf6 and ARNO (an Arf6 GTP/GDP exchange factor) recruitment to endosomal membranes. We propose that acidification-mediated vesicle coat assembly involves a complex "translocation machinery" with interactions between cytosolic proteins and a pH-sensing transmembrane endosomal protein. Cytosolic and transmembrane proteins associated with the "translocation machinery" will be identified using a combination of vesicle fractionation, proteomic technology and immununocytochemistry. We will define structural motifs involved in this targeting process using recombinant mutant protein constructs: 2) To elucidate the role of intra-vesicular acidification, GTPases and their regulatory proteins in endocytosis and transcytosis pathways in kidney epithelial cells in culture. We will study the role of the "translocation machinery" (ARNO, Arf6, Arf1) and phospholipases (PLD2, cPLA2) on protein reabsorption (endocytosis and transcytosis) by transfected kidney proximal tubule epithelial cells using established assays for these trafficking processes. The role of heterotrimeric GTPases (Gai2, Gai3) and their regulatory proteins (RGS2, RGS4, RGS-GAIP) endocytosis and transcytosis will be examined in these cells. The role of intra-vesicular acidification and the "acidification machinery" (V-ATPase and CLC5 Cl- channels) on protein reabsorption (endocytosis and transcytosis) will be investigated: 3) To use animal models (ClC5 knockdown and knockout mice, cadmium-induced Fanconi syndrome), to determine the functional consequences of intra-vesicular acidification defects and the role of GTPases in the pathogenesis of Dent's disease and Fanconi syndrome. This will be achieved using isolated endosomes from proximal tubules, and using primary cell cultures. These aims will be achieved using a combination of approaches from in vitro assays with recombinant GTPases and accessory proteins, proteomic analysis of endosomal proteins and their binding partners, cell cultures transfected with wild type and mutant proteins, and finally whole animal models in which endosomal acidification is impaired.